The present invention concerns a compound and a pharmaceutical composition for the treatment of inflammation and diseases accompanied by inflammatory processes, in particular inflammatory processes which affect cellular membranes. The present invention also concerns therapeutic methods to ameliorate or prevent symptoms of inflammatory processes.
Inflammatory Processes
Inflammation is generally accompanied by changes in the metabolism of arachidonic acid, metabolism of nitric oxide, and creation of free radicals. Anti-inflammatory non-steroid drugs (NSAIDS), such as aspirin, can block certain links of an inflammatory process, but these drugs cannot stabilize damaged cellular membranes, which makes their influence on an inflammatory process limited and insufficient.
Inflammation is a localized reaction of live tissue due to an injury, which may be caused by various endogenous and exogenous factors. The exogenous factors include physical, chemical, and biological factors. The endogenous factors include inflammatory mediators, antigens, and antibodies. Endogenous factors often develop under the influence of an exogenous damage. An inflammatory reaction is inevitably followed by an altered structure and penetrability of the cellular membrane. At the tissue and organ level, inflammation is indicated by pain, swelling, reddening, increased temperature, and a lost function in some cases. Inflammation begins with a sublethal damage and terminates either with a complete recovery or long-term tissue ruination. There is no recovery from an injury without an inflammation.
An immediate response to a tissue damage is realized via mediators, which are released due to the exocytosis or lysis of cells. The main inflammatory mediators are compounds of the kinine and fibrinolytic systems, the complement system, metabolites of arachidonic acid, vasoactive amines, and other chemical compounds. The chemical mediators of inflammation include: histamine, serotonin, prostaglandins, CGRP, nitric oxide, among others.
An important role in inflammations is played by various reactive oxygen-containing species. These compounds are synthesized when oxygen transforms them into very dangerous forms, producing free radicals, which are atoms and molecules with unpaired electrons. Different free radicals have different activity levels.
The launch of an inflammation is influenced by various exogenous and endogenous agents. Endogenous factors, namely, mediators, antigens, and autogens define the nature and type of the inflammatory reaction, especially its course in the zone of injury. In the case where a tissue damage is limited to the creation of mediators, an acute form of inflammation develops. If immunologic reactions are also involved in the process, through the interaction of antigens, antibodies, and autoantigens, a long-term inflammatory process will develop. Various exogenous agents, for example, infection, injury, radiation, also provide the course of inflammatory process on a molecular level by damaging cellular membranes which initiate biochemical reactions.
Inflammatory processes rely on the metabolism of arachidonic acid, which converts to prostaglandines (PG), tromboxanes (TX), and leukotrienes (LT). Prostaglandines, tromboxanes, and leukotrienes are the main participants of all inflammatory processes. There are two known ways of arachidonic acid cascade. The first way leads to the creation of prostaglandines G2 and H2. This process is catalyzed by prostaglandin-cyclooxygenase. Cyclooxygenase catalyzes the production of PGA2, PGE2, PGD2, PGF2xcex1, while tromboxane-synthesis with PGH2 produces tromboxane A2 (TXA2).
The cascade of metamorphoses of arachidonic acid, which is a product of membrane and phospholypase A2, is best known. Through its cyclogenase and lypoxygenase cascades, arachidonic acid turns into prostaglandins and leukotrienes, respectively. The cyclooxygenase way leads to the formation of two bio-active products: prostacycline (PGI2) and thromboxane (TXA2). These products are involved in many inflammatory effects: bronchoconstriction, vazodilation, vasoconstriction, platelet aggregation, analgesia, pyrexia, et al.
Another way of arachidonic acid metabolism with 5-lipoxygenase leads to the synthesis of leukotrienes: LTA4, LTB4, LTC4, LTD4, LTE4, and LTF4. These leukotrienes have a powerful anti-inflammatory and bronchoconstrictor action, and they play and important role in vascular penetrability. Besides, leukotrienes are known as potential chemotactic factors; they increase the migration of WBC and have a great influence on the slow-releasing substance of anafilaxis (SRS-A).
Prostaglandines can play an important role in the development of systemic inflammatory reactions. In rheumatic arthritis, large quantities of PG and LT in the synovial liquid support the development of an inflammatory process and demineralization of bone tissue surrounding joints. Leukotrienes are known to be the main patho-physiological mediators of inflammatory reactions. They influence, to a greater degree than prostaglandines, the penetrability of vessels and the adhesion of leukocytes to vessel walls as well as the development of edema.
Prostaglandines effectively regulate the aggregation of platelets. PGE1 is a powerful inhibitor of platelets aggregation, while PGE2, which is normally released from platelets, stimulates this process. However, the most important role in blood coagulability is played by PGI2, or prostacycline, which is synthesized in blood vessel walls by arachidonic acid. It is the most powerful inhibitor of platelets aggregation, which has vasodilator properties. Thromboxane, which is synthesized in platelets, has an opposite action.
When endothelium is damaged, the adhesion of platelets with subendothelium tissue and the aggregation of platelets is initiated. The main role in this process is played by thromboxane A2. Prostaglandin I2, on the contrary, inhibits the aggregation of platelets. Therefore, the proportion of PGI2 and TXA2 is crucial for the process of coagulation.
Further, a special role in the process of recovery from inflammation is played by nitrogen oxide (NO). This gas easily penetrates in different organs and tissues and, as a free radical, has a powerful reactivity. Nitrogen oxide is a potent vasodilator, neurotransmitter, and inflammatory mediator, which plays a significant role in asthmatic inflammation.
Nitrogen oxide is produced endogenously by L-arginine amino acid and NO-synthetase. There are three known forms of NO-synthetase, two of which are constituent, and one inducible. The inducible NO-synthetase, which is expressed in the epithelium cells, quickly increases its activity when anti-inflammatory cytokines (such as interleukin 1 beta (IL-1beta) and tumor necrosis factor (TNF-alfa) are released.
Nitrogen oxide has both positive and negative properties with respect to an inflammatory reaction. One important and potentially positive property is its ability to relax the smooth bronchial muscle, which results in bronchodilation. Its negative properties include the ability to help the inflammatory process by increasing chemotaxis neutrophils, monocytes, and oesinofils with the help of the guasine-monophosphate-dependent mechanism. It is believed that nitrogen oxide inhibits adhesion of leukocytes to vascular endothelium and bronchial epithelium.
NO plays an important biological role in defining basal vascular tonus, regulating contractions of myocardium, and modulating the interaction between thrombocytes and vascular walls (Zhou Q., Hellermann G. R., Solomonson L. P., Nitric oxide release from resting human platelets, Thromb.Res., 1;77(1):87-86; 1995). The role of thrombocyte activation in the pathogenesis of various thrombo-vascular conditions in humans and evidence about decreased NO-mediated effects in hypertension (Calver A., Collier J., Moncada S., Vallance P., Effect of local intra-arterial NG-monomethyl-L-arginin in patients with hypertension: the nitric oxide dilator mechanism appears abnormal, J.Hypertens., 10(9):1025-1031; 1990), diabetes (Calver A., Collier J., Valance P., Inhibition and stimulation of nitric oxide synthesis in the human foream arterial bed of patients with insulin-dependent diabet, J.Clin.Invest, 90(6):2548-2554; 1992), and artherosclerosis (Drexler H., Zeiher A. M., Meinzer K., Just H., Correction of endotelial dysfunction in coronary microcirculation of hypercholesterolaemic patient by L-arginine, Lancet., 21-28; 338(8782-8783); 1546-1550; 1991) suggests that drugs which increase the activity of NO-synthetase may effectively be used in treatment of patients. Human thrombocytes are capable of synthesizing nitric oxide. Large quantities of nitric oxide, for example, in the cells of endothelium, may be produced by intact thrombocytes, as well as by stimulated thrombocytes. Hence, nitric oxide of thrombocyte origin plays an important role in the support of vascular homeostasis and other NO-sensitive processes. (Zhou et al.,1995).
Beside some common features, inflammatory processes in each individual case have certain distinctions related to the peculiarities of functioning of the body organ and to the factors which caused the impairment: i.e., viruses, microorganisms, injuries, poisoning, etc.
For example, one of the common mechanisms of heart diseases, including acute infarct myocarditis, is a malfunction of the structure and function of the membrane of heart cells. As a result, the synthesis of leukotrienes, tromboxanes, etc., which have coronoconstrictor, arrythmogenic, hemoatractive and pro-aggregate action, increases (Bangham A. D., Hill M. W., Miller N., Preparation and use of liposom as model of biological membranes, Methods in Membrane Biology, Acad. Press, V.1, N. Y., P.1-68, 1974).
Another important factor in the pathogenesis of heart impairments is the coronoconstrictor and hemoattractive (with regard to neutrofiles) action of lipoxygenase derivatives LTC4, LTD4, LTB4 (Hoshida S, Kuzuya T., Nishida M., et al., Amer.J.Cardiol, 7; 63(10): 24E-2E; 1989; Lam B. K., Gagnon L., Austen K. F. et al., J.Biol.Chem., 15; 265(23): 13438-1341; 1990; Svendsen J. N., Hansen P. R., Ali S. et al., Cardiovasc.Res., 27(7): 1288-1294; 1993). Substances which can block this process can in turn reduce the size of necrosis at acute myocardial infarction and, therefore, significantly decrease the lethality in difficult cases of heart disease, such as gross myocardial infarction. At the same time, such substances can stabilize the membranes of heart cells. In addition, it is known that coronoconstrictor and hemoattractive effects during infarct are accompanied by an increased aggregation of platelets. Therefore, blocking this process also leads to a decrease of the size of impairment.
Further, disorders of the aggregate state of blood play an important role in the pathogenesis of various diseases. This is especially apparent in the pathogenesis of thrombo-vascular conditions in humans. It is known that a malfunction in the thrombo-vascular link of homeostasis is a key factor leading to disorders of the aggregate state of blood, by causing changes in the rheological properties of blood and triggering the formation of internal vascular aggregates. Thrombocyte-related injuries lead to failures in micro-circulation processes, which result in shortages of blood inflow to the tissue. At the initial stage of the formation of blood clots, platelets become activated and further undergo adhesion to the injured endothelium. Later on, they aggregate and an initial thrombocytic blood clot is formed.
Today, there is enough evidence of a close relation between inflammations, disorders in the aggregate state of blood, and cardio-vascular conditions (Anderson J. L. Cariqist J. L., et al., Evaluation of C-reactive protein an inflammatory marker, and infectious serology as risk factors for coronary artery disease and myocardial infarction, J.Am. Coll. Card., 32: 35-41; 1998).
Role of Cell Membranes in Inflammatory Processes
The functions of cell membranes and their relation to inflammatory processes has been documented. It is known that the plasmatic cellular membrane occupies a special place among the other membrane structures and performs such important functions as barrier and transportation, provides a contact with the outside environment for the cell, participates in the regulation of cellular homeostasis, supports signal mechanisms of this regulation, and defines the cell""s individuality and wholeness. The structural organization, dynamics, and functions of erythrocytal membranes and various hemolysis patterns, such as osmotic, oxide, immune (induced by hemolytic viruses, toxins, complement), detergent hemolysis, photohemolysis, etc., are well studied (see, e.g., Bashford C. L., Alder G. M., Menestrina G.,et al., Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cation. J. Biol. Chem., 15; 261(20): 9300-9308, 1986; Osorie e Castro V. R., Ashwood E. R., Wood S. G., Vernon L. P., Hemolysis of erythrocytes and fluorescence polarization changes elicited by peptide toxins, aliphatic alcohols, related glycols and benzylidene derivatives, Biochim. Biophys. Acta., 16; 1029(2): 252-258; 1990).
It was demonstrated that pH variation in the outside environment upsets the balance of forces influencing the membrane, which leads to structural changes and changes of the aggregation degree of membrane proteins. Two types of membrane structural changes are distinguished: those caused by pH variation in the range 7.0-6.0, and those for pH levels below 4.5 (Zavodnik I. B., Pileckaya T. P., Acid lysis of human erythrocytes, Biophizika., V.42, N.5, P.1106-1112, 1997). In the latter case, the membrane becomes destabilized and erythrocytal lysis follows. It is known that at pH 4.7, pores are formed in glycocalyx erythrocytal membranes (Arvinte T., Cudd A., Schulz B., Nicolau C., Biochim. Biophys. Acta., 19; 981(1): 61; 1989). In particular, decreased pH levels of the environment change the confirmation, package type, and mobility of phospholipids in model membranes. Thus, aggregation of membrane proteins, denatured due to a decreased pH, is the reason for membrane damages and acid lysis in erythrocytes.
The pattern of erythrocytal hemolysis by HCl was proposed based on the cooperative protonation of some center located in stroma or on the membrane of erythrocyte with a subsequent creation of pores, sufficient to release hemoglobin. By studying the mechanism and pattern of the acid hemolysis process, information about the structural organization of the membrane and membrane-stabilizing actions can be obtained.
The best known endogenous stabilizers of hemolysis in erythrocytes (osmotic hemolysis is the best-studied) are albumin of blood plasma, metallic ions K+, Na+, Mg2+ and, especially, Ca2+, which modulate the canals of plasmatic erythrocytal membranes, possibly including the proton canal (Anderson D. R., Davis J. L., Carraway K. L., Calcium-promoted changes of the human erythrocyte membrane. Involvement of spectrin, transglutaminase, and a membrane-bound protease. J. Biol. Chem., 10; 252(19): 6617-6623, 1977), cholesterol adsorbed on the surface of erythrocytes (Hui S. W., Stewart C. M., Carpenter M. P., Stewart T. P. Effects of cholesterol on lipid organization in human erythrocyte membrane, J. Cell. Biol., 85(2): 283-291; 1980), and polyamines, which bind with the fatty-acid residues of membrane phospholipids (Rennert O. M., Shukla J. B., Polyamines in health and disease Advances in Polyamine research, Raven Press, V.2, N.Y, P.195-21, 1978). The best known activators of endogenous hemolysis in erythrocytes are long-chain fatty acids (Rybszynska M., Csordas A, Chain length-dependent interaction of free fatty acids with the erythrocyte membrane, Life Sci., 44(9): 625-682; 1989), and especially free radicals of oxygen and nitrogen (Sato Y., Kamato S., Takahashi T. et al., Mechanism of free radical-induced hemolysis of human erythrocytes: hemolysis by water-soluble radical initiator. 18; 34(28): 8940-8949; 1955; Sen T.,Ghosh T. K., Chaudhuri A. K. Glucose oxidase-induced lysis of erythrocytes. J,Exp.Biol., 33;(1): 75-76; 1995; Wollny T., Yacoviello L. Propogation of bleeding time by acute hemolysis in rats: a role for nitric oxide. Am. J. Physiol. 272(6): 2875-2884; 1997).
In summary, there is evidence to suggest that the structure of the membrane is altered during inflammatory processes. However, the model of membrane damage in the inflammatory process has not been used for screening drugs and treating or preventing inflammation and inflammatory-related disorders.
Present Drugs Unsatisfactory
The present anti-inflammatory drugs are unsatisfactory because the difficult and various biochemical reactions involved in inflammations and the lack of reliable information about inflammatory pathogenesis complicate the experimental choice of pharmacological compounds capable to regulate inflammation. Thus, drugs are selected to have an effect on individual components of an inflammation. So far, there is no drug able to regulate most of the components of any inflammatory reaction.
Most of the known non-steroid anti-inflammatory drugs (NSAIDS) selectively influence certain phases of this pathological process. First, they influence the penetrability of blood vessels, which is often altered in acute inflammations, and various cell reactions, which are common for chronic inflammations. Also, many NSAIDS influence metabolism through the mechanism of free radicals.
The initial screening of anti-inflammation processes typically uses three groups of methods. First, the influence of drugs on easily-identifiable inflammatory symptoms is studied. These include swelling, hyperemia, necrosis, etc. A more advanced analysis includes experimental therapy methods, using model arthritis, carditis, etc., which are similar to human ailments. The third stage involves analysis of how the drug influences certain metabolic ways.
After the metabolism of arachidonic acid was studied in detail, many anti-inflammatory compounds, whose action was to regulate the formation of such metabolic products, were proposed. In most cases, such drugs act as inhibitors of the metabolic enzymes of arachidonic acid. One example is the anti-inflammatory pharmacological combination of cyclooxygenase 2 inhibitor and leukotriene A.sub.4 hydrolase inhibitor (Isakson, P. C., Anderson G. D., Gregory, S. A., Treatment of inflammation and inflammation-related disorders with a combination of a cyclooxygenase-2 inhibitor and a leukotriene A.sub.4 hydrolase inhibitor, U.S. Pat. No. 5,990,148, November 1999). A similar approach was proposed on the basis of analogues of pyrimidines, a component of nucleic acids (Connor D. T., Kostlan C. R., Unangst P.C., 2-heterocyclic-5-hydroxy-1,3-pyrimidines useful as antiinflammatory agents, U.S. Pat. No. 5,240,929, August 1993). Since these compounds are the inhibitors of key metabolic ferments of arachidonic acid, 5-lipoxygenase and cyclooxygenase, the authors suggested their use as anti-inflammatory drugs suitable for treatment of a wide range of diseases, from allergenic conditions and rheumatoid arthritis to artherosclerosis and myocardial infarction. Other researchers recommended prostacyclin analogues for treatment of thrombocyte aggregation and bronchoconstriction (Haslanger M. F., Prostacyclin analogs and their use in inhibition of arachidonic acid-induced platelet aggregation and bronchoconstriction, U.S. Pat. No. 4,192,891, March 1980).
However, since an inflammatory process initiates many different metabolic cascades, the use of inhibitors or metabolic analogues of arachidonic acid does not allow to balance all such reactions and, hence, cannot regulate the complex inflammatory process in a satisfactory manner.
Further, aspirin, which has been used in applied medicine for a long time, has also been proposed since it can block metabolic ferments of arachidonic acid. Inhibitors of prostaglandines, such as aspirin, quite effectively influence the inflammatory processes. For this reason, they are successfully used in clinics for the treatment of rheumatoid arthritis, osteoarthritis, and other similar inflammatory processes. Aspirin also has anti-coagulation properties, since it inhibits the synthesis of TXA2, and it influences at least partially the synthesis of PGI2. A daily dose of 3 g of aspirin is commonly used for prevention of stenocardia, as a post-infarct and post-insult treatment, or for patients with a high risk of cardio-vascular conditions.
However, studies on the synthesis of TXA2 and PGI2 in vivo have shown that peroral administration of aspirin decreases the secretion of PGI2 only for 2-3 hours, while the secretion of thromboxane is halted for 10 days (Vesterqvist O., Measurements of the in vivo synthesis of thromboxane and prostacyclin in humans, Scand. J. Clin. Lab. Invest. 48(5): 401-407; 1988). This author, as well as others (see, e.g., Lorenz R. L., Boehlin B., Uedelhoven M. W., Weber P. C., Superior antiplatelet action of alternate day pulsed dosing versus split dose administration of aspirin, Am. J. Cardiol. 15; 64(18): 1185-1188; 1989), not only show the difficulties in administering the right dose of aspirin, but also provide and experimental ground for the frequent side effects caused by aspirin during its long-term use.
Specifically, aspirin and other non-steroid anti-inflammatory drugs may be the cause of anaphylactoid reactions in sensitive individuals. The mechanism of these reactions is dose-dependent toxic-idiosyncratic, not immunologic. Also, aspirin is the most common cause of accidental poisoning. Children, treated by aspirin before poisoning, are also at great risk. Aspirin overdose, which occurs frequently, is difficult to correct. The effective aspirin dose for many diseases, including rheumatoid arthrtis, constitutes 3-6.5 mg per day, which leads to irritations of the gastro-intestinal tract. Patients with gastro-intestinal conditions do not tolerate aspirin. Aspirin also causes erosion, bleeding stomach ulcers, diarrhea, and duodenum ulcers. Further, aspirin is commonly used in treatment for its anti-thrombocytic action, but it is badly tolerated and causes side-effects when taken for a long period of time. In addition, by inhibiting non-selectively cyclooxygenesis, aspirin interferes with the synthesis of thromboxane, which is a powerful aggregant and vasoconstrictor, and may also lead to decreased levels of prostacycline, which is both anti-aggregant and vasodilator.
All these negative side-effects of aspirin and other NSAIDS motivate the search for new drugs which would have anti-inflammatory properties, but which are non-toxic in a wide range of concentration, have no side effects during a long-term use, and are capable of preventing and terminating inflammatory processes.
Pharmaceutical Use of Nucleic Acids
Nucleic acids are commonly used in pharmacology (Rothenberg M., Jonson G., Laughlin C. et al. Oligodeoxynucleotides as anti-sense inhibitors of gene expression: therapeutic implications, J. Natl. Cancer Inst., 18; 81(20): 1539-1544; 1989; Zon G., Oligonucleotides analogues as potential chemotherapeutic agents, Pharm. Res., 5; (9): 539-549; 1988). However, pharmaceutical uses for nucleic acids have not included inflammatory or inflammatory-related disorders. For example, Anderson et al., proposes the method of modulating the effects of cytomegalovirus infections with the help of an oligonucleotide, which binds with mRNA of cytomegalovirus, for treatment of cytomegalovirus infections in humans (Anderson K., Draper K., Baker B., Oligonucleotides for modulating the effects of cytomegalovirus infections, U.S. Pat. No. 5,442,049, Aug. 15, 1995). On the basis of a specific nucleic acid, which encodes the succession of 3xe2x80x2non-translated sector of protein kinase C, Boggs et al. propose a method for diagnosis and treatment of conditions, which are associated with protein kinase C alpha (Boggs R. T., Dean. N. M., Nucleic acid sequences encoding protein kinase C and antisense inhibition of expression thereof, U.S. Pat. No. 5,681,747, October 1997). Also, Yano et al. patented a DNA compound obtained from Mycobouterium bovis and Bacillus subtilis for treatment of stomach ulcers (Yano O., Kitano T., Method for the treatment of digestive ulcers, U.S. Pat. No. 4,657,896, April 1987).
In particular, it is known that ribonucleic acid (RNA), products of its partial hydrolysis, and synthetic poly-ribonucleotides have a wide range of bioactivity (Kordyum V. A., Kirilova V. S., Likhachova L. I., Biological action of exogenous nucleic acids, Visnyk ASC USSR, V.41, N.6, P.67-78, 1977). They activate protein synthesis in cells (Sved S. C., The metabolism of exogenous ribonucleic acids injected into mice, Canad.J.Biochem.,V.43, N.7, P.949, 1965) and have anti-tumor activity (Niu M. C., Effect of ribonucleic acid on mouse acids cells, Sciens., N.131, P.1321, 1960). RNA can increase antibody generation and decrease the inductive phase of antibody genesis (Johnson A. G., Schmidtke I., Merrit K. et at., Enhancement of antibody formation by nucleic acids and their derivatives, in Nucleic acid in immunology, Berlin, P.379, 1968; Merrit K., Johnson A. G., Studies on the adjuvant of bacterial endotoxins on antibody formation, 6. Enhancement of antibody formation by nucleic acids, J.Immunol., V.94, N.3, P.416, 1965; Brown W., Nakono M., Influence of oligodeoxyribonucleotides on early events in antibody formation, Proc. Soc. Exper. Biol. Med., 5, V.119, N.3, P.701, 1967). It was shown that certain increased or decreased immunologic indicators normalize under the influence of RNA. In the first place, this applies to T-lymphocytes, cooperation of T- and B-lymphocytes, activation of macrophage function, etc.
Further, exogenous RNA is used for the DNA synthesis in dividing cells and for the RNA synthesis in metabolizing cells. It was also determined that 2 hours after the introduction, exogenous RNA was included in the RNA of lymphocytes and macrophages (Enesco N. E., Fate of 14C-RNA infected into mice, Exper. Cell Res., V.42, N.3, P.640, 1966). Evidence suggests that yeast tRNA can be included into cells in the form of intact molecules (Herrera F., Adamson R. H., Gallo R. C., Uptake of transfer ribonucleic acid by normal and leucemic cells, Proc.Nat.Acad.Sci.USA, 67(4): 1943-1950; 1970).
It was determined by analytical methods that RNA is present in practically all membranes of animal cells (membranes of endoplasmic reticulum, mitochondrial, nucleic, and plasmatic membranes). Its content, depending on the type of tissue and on the method of membrane isolation, varies between 0.5 and 4% of the dry weight of the membrane. Experimental results show that special membrane RNA exists in isolated membranes (Shapot V. S., Davidova S. Y., Liponucleoprotein as an integral part of animal cell membrans. Prog. Nucleic Acid Res. 11: 81-101; 1971; Rodionova N. P., Shapot V. S. Ribonucleic acid of the endoplasmatic reticulum of animal cells. Biochim et Biophis Acta, 24; 129(1); 206-209; 1966). The functions of membrane RNA are not fully understood.
The functions of membrane RNA in ribosome have been better studied. (Cundliffe E., Intracellular distribution of ribosoms and poliribosomes in Bacillus megaterriium. J.Mol.Biol., 28; 52(3): 467-481; 1970) Ribosomal RNA is contained in bacterial membranes, in the outer membranes of nuclei, inner and outer membranes of mitochondria, inner membrane of the Goldgi apparatus, which adjoins the plasmatic membrane, in the rugged endoplasmic reticulum, in different tissues in animals, humans, plants, microorganisms, and protozoa. It is possible that membrane glycolipids and glycoproteins, which contain N-acetylneuraminic acid, are involved in the formation of binding sites of ribosomal RNA in ribosomes, since membranes which are treated by neuronidase lose the ability to bind ribosomes. (Scott-Burden T., Hawtrey A. O., Preparation of ribosome free membranes from rat liver microsomes by means of lithium chloride. Biochem. J. 115(5): 1063-1069; 1969. Further, it is possible that binding sites of ribosomes and membranes are activated by the sexual hormones, and cancerogens damage this physiological mechanism. This conclusion is supported by decreased levels of membrane-bound RNA in the process of aging (Mainwaring W. J. The effect of age on protein synthesis in mouse liver. Biochem J. 113(5): 869-878; 1969) and after castration of animals (Tata J. R., The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis. Biochem J. 105(2): 783-801, 1967). Extraction of spermine from a membrane leads to a separation of bound RNA from the membrane (Khawaja J. A. Interaction of ribosomes and ribosomal subparticles with endoplasmic reticulum membranes in vitro: effect of spermine and magnesium. Biochim. Biophis. Acta., 29; 254(1): 117-128); 1971). When membranes are treated with RNA of native small ribosomes of myeloma cells, they separate from the membranes, while large native subunits of ribosomes remain bound with the membranes (Mechler B.,Vassalli P., Membrane-bound ribosomes of myeloma cells.I.Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of ribosomes. J.Cell.Biol. 67(1): 1-15; 1975 . Also, the nucleotide components of various membrane enzymes, for example, polyA-RNA enzyme of phosphofructokinase, constitute a possible pool of membrane RNA (Hofer H. W., Pette D. The complex nature of phosphofructokinasexe2x80x94a nucleic acid countaining enzyme, Life Sci. 4(16): 1591-1596; 1965).
However, nucleic acids, and in particular RNA, and compositions containing the same, have not been used to treat or prevent inflammatory or inflammatory-related disorders. In particular, most of the studies above rely on experiments in vitro. Further, none of these methods is directed to treating or preventing an inflammation or inflammatory-related disorder.
Need for New Drug
In view of the above, there is a need for new anti-inflammatory drugs which would regulate disorders of the aggregate state of blood and would have less negative effects than aspirin and other NSAIDS. In particular, since an inflammatory process in the initial stage is followed by alterations in the structure and functions of the membrane in the many cells involved in the inflammatory process, drugs are needed which, not only regulate all the components of an inflammatory metabolic cascade, but also stabilize membrane structures and functions in the involved cells. In particular, since the traditional therapy has little effectiveness in extensive infarcts, which are complicated by the cardiogen shock, there is a need for new drugs capable of stopping the destruction of cardiomyocytes.
The present invention offers a compound, a pharmaceutical composition and a method for the treatment or prevention of inflammation and diseases accompanied by inflammatory processes. The compound is an active ingredient consisting of RNA, in particular RNA extracted from yeast. Yeast RNA is a heterogenous compound of low-polymeric RNA, which comprises various quantities of nucleotides, nucleotide polymers, and usually 5 to 25 nucleotides. Oligonucleotides and transport RNA with a great number of minor bases prevail in yeast RNA.
Since one of the common features of all inflammatory processes at a molecular level is altered penetrability and structure of membrane, the present invention was made using a method of selecting drugs based on their ability to stabilize cellular membrane in inflammations. Thus, by analyzing destructive mechanisms induced by various factors in plasmatic membranes and learning about the structural elements of their interaction, which provide the optimal organization of a cell, it is possible to select drugs having membrane-stabilizing action for applied medicine. Specifically, it has now been established that, since membranes contain low-molecular RNA which probably plays a membrane-stabilizing role, introduction into the body of exogenous low-molecular RNA leads to stabilization of disturbed membranes, such as, for example, membranes of cells involved in inflammatory processes.
Stabilization of the cell membrane by the compound of the present invention leads to the normalization of arachidonic acid metabolism and nitric oxide metabolism, which have a powerful anti-inflammatory action and are the main participants of all inflammatory processes, for example, rheumatoid arthritis, osteoarthritis, allergies (such as asthma), and other inflammatory conditions, such as pain, swelling, fever, psoriasis, inflammatory bowel disease, gastrointestinal ulcers, cardiovascular conditions, including ischemic heart disease and atherosclerosis, partial brain damage caused by stroke, skin conditions (eczema, sunburn, acne), leukotriene-mediated inflammatory diseases of lungs, kidneys, gastrointestinal tract, skin, prostatitis, and paradontosis.
The yeast RNA is effective in decreasing the activity of iNOS in the course of an auto-immune process, both during its initiation and in the chronic stage. This property allows the usage of yeast RNA in pathological conditions which are accompanied by iNOS induction: diabetes, tumor, hepatitis, infections, neuro-degenerate diseases (Parkinson""s disease, Alzheimer""s disease, multiple sclerosis, encephalitis), and others.
In addition, the use of natural molecules of nucleic acids, such as the compound of the present invention, in large concentrations as pharmacological compounds causes no or little side effects, especially taking into account the fact that this compound constantly enters human and animal bodies with food.
Further, the present invention offers a method for the treatment of inflammation or inflammatory-related disorder comprising administering to a mammal in need of such treatment an amount effective to ameliorate the symptoms of inflammation or inflammatory-related disorder of ribonucleic acid and a pharmaceutically acceptable vehicle, carrier, or diluent.
Still further, the present invention offers a method of stabilizing damaged cellular membranes which comprises administering to a mammal having damaged cellular membranes an amount effective to stabilize said damaged cellular membranes of ribonucleic acid and a pharmaceutically acceptable vehicle, carrier, or diluent.
Still further, the present invention offers a method of normalization of NO-synthetase ability in a mammal which comprises administering to a mammal in need of such treatment an amount effective to normalize NO-synthetase ability in the mammal of ribonucleic acid and a pharmaceutically acceptable vehicle, carrier, or diluent.
Still further, the present invention offers a method of inhibiting oxidation of components of cell membranes of a mammal, which comprises administering to a mammal in need of such treatment an amount effective to inhibit oxidation of components of cell membranes of the mammal of ribonucleic acid and a pharmaceutically acceptable vehicle, carrier, or diluent.
Still further, the present invention offers a method of inhibiting thrombocyte aggregation, which comprises administering to a mammal in need of such treatment an amount effective to inhibit thrombocyte aggregation of ribonucleic acid and a pharmaceutically acceptable vehicle, carrier, or diluent.
Also, the present invention offers a compound consisting of ribonucleic acid extracted from yeast, for example a Saccharomyces cerevisiae or a Candida utilis. Preferably, the ribonucleic acid has a nitrogen content of more than 14.5% by weight and a phosphorus content of more than 8.5% by weight, more preferably a nitrogen content of more than 14.7% by weight and a phosphorus content of more than 8.6% by weight, even more preferably a nitrogen content of more than 15.0% by weight and a phosphorus content of more than 9.0% by weight.
Further, the present invention offers a pharmaceutical composition for the treatment or the prevention of inflammation or inflammatory-related disorder, comprising ribonucleic acid and a pharmaceutically acceptable vehicle, carrier, or diluent.
A complex analysis of known nucleic acids was carried out using various in vitro and in vivo models. The models were chosen to correspond to certain types of inflammatory processes, both of common and immunologic origin. In the tests, the effects of ribonucleic acid (RNA), in particular yeast RNA, was compared to the effects of existing anti-inflammatory drugs over a wide range of anti-inflammatory activities.
Summary of Experimental Models and Results
1. Model of Thrombocyte Aggregation in Vitro
An initial screening of exogenous nucleic acids was conducted in vitro on the model of aggregation of human thrombocytes induced by arachidonic acid (Born L. V. R. The aggregation of blood platelets by difosfate and its reversal, Nature, V.94, P.327, 1962). Exogenous DNA and RNA from prokaryotes and eukaryotes were analyzed. We used aspirin as representative of a standard anti-inflammatory drug.
It was demonstrated that aspirin inhibited the aggregation of thrombocytes induced by arachidonic acid to a certain level. Desoxyribonucleic acid obtained from chicken erythrocytes (DNA-CE) produced by xe2x80x9cReanalxe2x80x9d (Hungary), inhibited thrombocytic aggragation within the range of aspirin. Further, DNA from cattle thymus (DNA-CT) produced by xe2x80x9cReanalxe2x80x9d (Hungary), and transport RNA of E. coli (tRNA) produced by xe2x80x9cServaxe2x80x9d (USA) inhibited aggregation of the induced thrombocytes almost twice. The highest inhibiting effect was demonstrated by total yeast RNA, which dramatically inhibited thrombocytic aggregation in a wide range of concentrations. Inhibition of thrombocytic aggregation by yeast RNA depended on the form (acid or its sodium salt), purity, and presence of protein. RNA-F with protein admixtures was less effective by a third. The sodium salt of yeast RNA-PN in high concentration was only half as effective, and did not act in low concentration.
Since the model of aggregation of thrombocytes induced by arachidonic acid is recognized for the selection of anti-inflammatory drugs, the results of this comparative test showed that nucleic acids, and especially RNA, in particular, yeast RNA, have pronounced anti-inflammatory properties.
2. Model of Acid Resistance of Erythrocyte Membranes in Vitro
Based on the recognition that destabilization of cellular membranes is the main indication of an inflammatory process, we used the model of acid resistance of erythrocyte membranes in vitro for the screening of membrane-protecting, and thus, anti-inflammatory properties of the drugs. We chose rat erythrocytes to study the immune-stabilizing action of exogenous nucleic acids. We analyzed the reactions of erythrocytic membranes to the destructive influence of nitric oxide. We estimated the membrane-stabilizing action of exogenous nucleic acids and damaging actions of endogenous and exogenous nitrite anion by calculating the acid resistance of erythrocytes according to the kinetic method (Terskov I. A., Hittelzon I. I., Chemical (acid) erythrogram method, Biophizika, 2(2): 259-266; 1957). The main idea of the method is to determine historical changes in the number of cells, which eventually become hemolyzed under the influence of weak acids. The lysis of erythrocytes in acid environment undergoes three stages: penetration of hydrogen ions (protons, H+) through the plasmatic membrane of erythrocytes, protonation of hemoglobin and membrane proteins, and, as a result, osmotic destruction of erythrocytes.
Using this method, we estimated the influence of exogenous nucleic acids on the kinetics of the penetration of protons through the erythrocytic plasmatic membrane, which depends on the membrane""s nature. The speed of proton penetration in the cellular cytosol depends to a great extend on the oxidation status of the lipid component (Kellogg E. W., Fridovich I., Liposome oxidation and erythrocyte lysis by enzymically generated superoxide and hydrogen peroxide J. Biol. Chem. 10; 252(19): 6721-4728; 1977) and protein component, especially, the band 3 oxidation of plasmatic membranes and is defined by the activity [H+]-ATP-ase, and the activity of various exchangers (Sato Y., Kamo S., Takahashi T., Suzuki Y., Mechanism of free radical-induced hemolysis of human erythrocytes: hemolysis by water-soluble radical initiator, Biochemistry, 18; 34(28): 8940-8949; 1995; Lukacs G. L., Kapus A., Nanda A. et al, Proton conductance of the plasma membrane: properties, regulation, and functional role, Am. J. Physiol, 265(1 Pt 1): C3-C14; 1993).
Acid erythrograms were recorded by the kinetic method. In the in vitro tests, acid erythrograms were recorded in the presence of sodium nitrite (the damaging agent) and different concentrations of exogenous nucleic acids.
The in vitro tests, which used the oxide damage model of erythrocytes by nitrite anion, a stable metabolite of nitric oxide, demonstrated stabilizing and membrane-protector action of exogenous nucleic acids.
On the model of acid resistance of erythrocytic membranes, we tested the same set of preparations as in the model of thrombocytic aggregation.
Yeast RNA preparations demonstrated membrane-protecting properties in a wide range of concentrations. A more detailed analysis showed that the membrane-protector action of yeast RNA depends on their form (acid or sodium salt), purity, and the presence of protein. Well-purified ribonucleic acid RNA-P, whose erythmograms in the concentrations 10 and 100 xcexckg corresponded to the norm, showed the highest effectiveness. Sodium salt of yeast RNA-PN was less effective, especially in the concentration 10 xcexckg. Protein admixtures in RNA-F resulted in a complete loss of the membrane-stabilizing action. Other preparations, tRNA, DNA-CT, and DNA-EC destabilized erythrocyte membranes at the tested concentrations, which means that they cannot be used as anti-inflammatory drugs as advantageously despite their anti-inflammatory properties demonstrated on other models.
3. Model of Erythrocytal Auto-Immune Reaction in Rats
We used the model of acid injury of erythrocytal plasmatic membranes to study the membrane-stabilizing action of exogenous nucleic acids. Acid damages to the protein and lipid components of erythrocytal plasmatic membranes were tested in vivo in the process of development of an auto-immune reaction (adjuvant arthritis). The biosynthesis of nitric oxide, which is an active oxidizing agent, became activated and, especially, hemoglobin of erythrocytes (Eich R. F., Li T., Lemon D. D. Mechanism of NO-induced oxidation of myoglobin and hemoglobin. Biochemistry, 4; 35(22): 6976-6983; 1966; Huot A. E., Kruszyna H., Kruszyna R. et al., Formation of nitric oxide hemoglobin in erythrocytes co-cultured with alveolar macrophages taken from bleomycin-treated rats. Biochem.-Biophys. Res. Commun., 15; 182(1); 151-158; 1992; Kosaka H., Harada N., Watanabe M. et al. Synergistic stimulation of nitric oxide hemoglobin production in rats by recombinant interleukin 1 and tumor necrosis factor. Biochem. Byophis. Res. Commun. 30; 189(1): 392-398; 1992). Nitric oxide, as well as hydrogen peroxide, plays a crucial role in the damage to cells, including blood cells, in the process of development of autoimmune reactions. The anti-inflammatory cytokines (gamma-interferon, IL-1) induce expression of the inducible isoform of NO-synthetase (iNOS).
We studied changes in the activity of NOS in rat blood in the development of autoimmune reaction (adjuvant arthritis) in order to evaluate the preparation""s immune-modulating effect and to obtain information about possible levels of one of the most active oxidizing hemolytics, nitric oxide (in the form of its stable metabolite, nitrite anion). We calculated the activity of the enzyme NO-synthetase (NOS), which generates endogenous nitrite anion. These values characterize the protective effect of exogenous nucleic acids against the damaging influence of nitrite anion on erythrocytic membranes. Our focus on the changes in stability of erythrocytes in the process of autoimmune reactions is due to the large existing body of evidence supporting the immune-modulating properties of erythrocytes (Karalnik B. V., Erythrocytes, their receptors, and immunity, Uspekhi Sovremennoy Biologii., V.112, N.1, P52-61, 1992; Prokopenko L. H., Siplivaya L. E., Erythrocytes as modulators of immunologic reactions, Uspekhi Phiziologicheskikh Nauk., V.23, N.4, P.89-106, 1992), which has resulted in the use of the term xe2x80x9cerythrocytal immune systemxe2x80x9d.
Development of the autoimmune process was accompanied by a substantial decrease of acid resistance of erythrocytes during the early stage and, on the contrary, by a considerable excess over the norm during the final stage, in comparison with the resistance of normal erythrocytes.
Yeast RNA increased membrane stability, i.e., normalized the process of transportation of protons (which is attributed to the state of the protein and lipid components of etrythrocytal plasmatic membranes) during the initial stage and kept it stable, close to the norm, during the following stages of autoimmune reaction.
Further, it was demonstrated that, during the development of an autoimmune process, activities of NOS in rat blood changed. During the initial and final stages, an increased activation of NOS in rat blood was evidenced. Yeast RNA decreased NOS activity, so that at the final stage, the activity was practically normal.
Also, development of the autoimmune process was accompanied by a substantial decrease of acid resistance of erythrocytes during the early stage and, on the contrary, by a considerable excess over the norm during the final stage, in comparison with the resistance of normal erythrocytes. Yeast RNA increased membrane stability during the initial stage, by normalizing the process of proton transportation, which is dependent on the state of the protein and lipid components of etrythrocytal plasmatic membranes, and kept it stable, close to the norm, during the following stages of autoimmune reaction.
In view of the above, the protecting activities of yeast RNA as shown on the model of autoimmune process establish its ability to cure, not only allergic diseases, but other chronic inflammatory processes as well, such as arthritis, artherosclerosis, and other diseases that involve autoimmune reactions. ps 4. Model of Swelling Induced by Carrageenan in Rats
To screen nucleic acid""s anti-inflammatory action, we used a common model of inflammatory swelling of leg in mice provoked by a sub-plantar injection of carrageenan. Carrageenan-induced swelling is sensitive to the action of compounds which reduce capillary penetrability.
During the initial stage, a significant role in the mechanism of anti-inflammatory effect of carragenan is played by kinine, while at the later stage, proteolytic ferments and prostaglandins become more important. The carrageenan model has a slower development and is preserved for a sufficient time, which makes it possible to study the biochemical mechanism of the anti-inflammatory action of a drug. Therefore, we used this model to study the influence of yeast RNA on the synthesis of thromboxane and leukotriene. At the same time, we analyzed the influence of yeast RNA on NO-synthetase activity.
Analysis of the anti-inflammatory action of nucleic acids in the carrageenan model showed that they all have certain anti-inflammatory action. However, only yeast RNA in the concentration 10 mg of drug per mouse resulted in a 50% reduction of swelling. The concentrations of yeast RNA tested in mice represented 1 to 15 mg per mouse. Concentrations below 1 mg of yeast RNA preparation per mouse did not show any action. In concentrations above 15 mg, reduction of swelling was about 53-55%. Further, biochemical tests revealed a stabilizing influence of yeast RNA on the activity of NO-synthetase as well as on the quantities of thromboxane and leukotriene, which varied in the course of swelling process.
By contrast, aspirin, which was tested at the recommended therapeutic dose of 20 mg/kg, influenced swelling to a considerably smaller extend and did not show stabilizing properties at the level of biochemical metabolism.
5. Model of Acute Ischemia in Rats
Further analysis of yeast RNA was conducted on the model of acute ischemia-reperfusion of myocardium in rats. This model is based on a common fundamental mechanism in the development of a variety of different heart conditions, which includes alteration of structures and functions of the membranes in endotheliocytes, cardiocytes, and other heart cells. This alteration results in the degradation of membrane phospholipids and the creation of highly effective bio-active compounds, such as leukotrienes or thromboxanes, which have coronaroconstrictor, arythmogen, chemoactive, and pro-aggregant action (Bangham A. D., Hill M. W., Miller N., Preparation and use of liposom as model of biological membranes, Method in Membrane Biology, Acad.Press, V.1, N.Y, P.1-16, 1974).
As the tests demonstrated, yeast RNA, injected in rats intravenously in the concentration of 40 mg per rat, normalized heart function in acute infarcts. This was shown in a pronounced anti-arythmic action of the compound and a substantial decrease of the necrosis area in ischemized myocardium of heart. The drug almost completely normalized NO-synthetase activity in blood and in the border zone of ischemized heart. Yeast RNA injection normalized to a certain level the content of arachidonic acid in blood and heart of animals in acute infarctions. The injection of yeast RNA almost completely normalized the levels of eukosanoids in rat blood in ischemia cases. The activity of mieloperoxidase, the marker enzyme of neutrophils which helps to evaluate the preparation""s anti-oxidant action, decreased almost twice in animals with infarct treated by yeast RNA.
The analysis of yeast RNA activity in the ischemia-reperfusion model in rats determined that the drug has a substantial stabilizing action in different cascades of inflammatory processes in the ischemized heart, which is expressed in its long-term anti-infarct action and a decreased size of the infarct area in myocardium.
On the basis of the study of yeast RNA action in ischemia-reperfusion of animal heart, we can conclude that yeast RNA has an anti-infarct action, or anti-inflammatory action in infarcts, through stabilization of the structure and function of membranes in endotheliocytes, cardiocytes, and other heart cells.
Experimental Procedures and Test Results